By analyzing the lifetime of the excited states of a sample labeled with one or more fluorescent dyes, important information can be obtained about the properties of the sample. Especially when multiple fluorescent dyes are used, information about a sample region being analyzed, such as information about the composition and surroundings thereof, can be obtained using fluorescence lifetime imaging microscopy (FLIM). In cell biology, for example, the calcium concentration in a sample region can be indirectly inferred by measuring the lifetime of the fluorescent dyes.
There are a number of methods for measuring the lifetime of the excited states of fluorescent dyes. Some of these methods are described in detail in Chapters 4 and 5 of the textbook by Joseph R. Lakowicz entitled “Principles of Fluorescence Spectroscopy,” Kluwer Academic/Plenum Publishers, 2nd ed., 1999. For example, it is possible to modulate the power of the excitation light over time so that conclusions about the lifetime of the excited state can be drawn from the phase delay of the emitted light.
It is also possible to excite the fluorescent dyes with short light pulses so that the time delay of the emission pulses can be measured electronically. German Patent Publication DE 10 2004 017 956 A1, for example, describes a microscope for analyzing the lifetime of excited states in a sample, which includes at least one light source for generating excitation light and at least one detector for receiving detection light emanating from the sample. The microscope is characterized in that the light source includes a semiconductor laser which emits pulsed excitation light, and that an adjusting device is provided for adjusting the pulse repetition rate to the specific lifetime properties of the sample.
In particular, the electronics required for data analysis is commercially available, often in the form of PC plug-in cards. However, apart from the high cost, such a time measurement card has the disadvantage of a very long dead time, so that, upon excitation of the sample, it can only detect the arrival of the first detection pulse (first detection photon) and is then “blind” for a significant period of time. Ultimately, a significant portion of the information contained in the detection light emanating from the sample remains hidden from the user.
Moreover, it is not possible to achieve a high repetition rate for the excitation pulses and, therefore, it is also not possible to perform frequent measurements within one measurement period. The actually achievable measurement rate is far below the usual repetition rates of commercially available pulsed lasers. For this reason, it usually takes a very long time until sufficient data is collected, for example, to generate a FLIM image.